Characterization of Beta-lactam Resistance in the Gastric Pathogen Helicobacter pylori

Helicobacter pylori is a major cause of peptic ulcer disease and potentially stomach cancer. Treatment of patients with gastritis is warranted and normally done with a combination of antibiotics (such as amoxicillin, clarithromycin or others) and a proton pump inhibitor. Resistance to most of these antibiotics in H. pylori has been seen worldwide and is constantly increasing.

The purpose of this thesis was to more systematically characterize the mechanisms of amoxicillin resistance in H. pylori. We used both a clinical isolate strain B258, the first US amoxicillin resistant strain to be examined in this way, and in vitro selected amoxicillin resistant strains. In strain B258 we determined that all

of the amoxicillin resistance was due to the presence of 2 amino acid substitutions in Penicillin Binding Protein 1(PBP1). To explain how these amino acids could increase amoxicillin resistance, we did homology modeling of PBP1 to map the location of these amino acids and their relation to the putative amoxicillin binding cleft.

To better appreciate the role of single amino acid substitutions in amoxicillin resistance, we made site-directed mutants of the most commonly found amino acids in PBP1 from amoxicillin-resistant strains. and transformed a sensitive strain with each to monitor its effect on amoxicillin resistance. We also used homology modeling and affinity binding studies using biotinylated amoxicillin to examine the role of these PBP1 mutations in amoxicillin resistance.

Finally, we examined a series of 6 in vitro selected amoxicillin resistant strains each with slightly increased MICs to amoxicillin, in order to track both the evolution of resistance as well as identify other possible novel resistance mechanisms. Whole genome sequencing of the most resistant isolate when compared to the parental sensitive strain, identified 12 genes as likely candidates affecting amoxicillin resistance. Changes in PBP1, PBP 2 and various outer membrane proteins were identified and their role in amoxicillin resistance is being systematically examined in each of the 6 isolates. Although studies are on-going, results to date have shown that in addition to PBP1, changes in outer membrane permeability are required to get high level amoxicillin resistance.